Hypothalamic-pituitary organoid generation through the recapitulation of organogenesis.

This paper overviews the development and differentiation of the hypothalamus and pituitary gland from embryonic stem (ES) and induced pluripotent stem (iPS) cells. It is important to replicate the developmental process in vivo to create specific cells/organoids from ES/iPS cells. We also introduce the latest findings and discuss future issues for clinical application. Neuroectodermal progenitors are induced from pluripotent stem cells by strictly removing exogenous patterning factors during the early differentiation period. The induced progenitors differentiate into rostral hypothalamic neurons, in particular magnocellular vasopressin+  neurons. In three-dimensional cultures, ES/iPS cells differentiate into hypothalamic neuroectoderm and nonneural head ectoderm adjacently. Rathke's pouch-like structures self-organize at the interface between the two layers and generate various endocrine cells, including corticotrophs and somatotrophs. Our next objective is to sophisticate our stepwise methodology to establish a novel transplantation treatment for hypopituitarism and apply it to developmental disease models.

The role of miR-7 as a potential switch in the mouse hypothalamus-pituitary-ovary axis through regulation of gonadotropins.

The hypothalamus-pituitary-ovary (HPO) axis plays fundamental roles in female neuroendocrinology and reproduction. Pituitary gonadotropins are located in the center of this axis. Previous investigation suggested that miR-7 is closely linked with gonadotropins. However, the interaction between miR-7 and the HPO axis remains unclear. This study aims to determine whether and how miR-7 functions in this axis. A mouse ovariectomy model and mouse primary pituitary cells were used in this study. The results showed that miR-7 is localized to gonadotrophs and somatotrophs. miR-7 can inhibit the expression, synthesis and secretion of gonadotropins, but not growth hormones. Gonadotropin-releasing hormone (GnRH) has inhibitory effects on miR-7, while estrogen enhances miR-7 expression. miR-7 is vital for the pathway by which GnRH and estrogen regulate gonadotropins by targeting v-raf-leukemia viral oncogene 1 (Raf1). Together, these results indicate that miR-7 acts as a potential switch in the feedback loop of the HPO axis by regulating gonadotropins.

Effects of Diets Enriched in Linseed and Fish Oil on the Expression Pattern of Toll-Like Receptors 4 and Proinflammatory Cytokines on Gonadal Axis and Reproductive Organs in Rabbit Buck.

Infections of the genital tract can perturb the fertility in humans and animals. Pathogen recognition and activation of innate immunity onset through the pattern recognition receptor activation, such as Toll-like receptor 4 (TLR4), leading to the production of proinflammatory cytokines and mediators. TLR4 is expressed both on leukocytes and nonimmune cells. Rabbit TLR4 shows great similarity to its human counterpart. Moreover, the TLR4 signalling pathway could be modulated by long-chain polyunsaturated fatty acids (LC-PUFA). The objectives of this study were (i) to determine the expression levels of TLR4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit and (ii) to evaluate if the n-3 PUFA-enriched diets can modify their expression levels in the tissues and LC-PUFA profiles in seminal plasma. Fifteen rabbit bucks (n = 5/experimental group) were fed with different diets: commercial standard (group C), rich in extruded linseed (10%, group L), and in fish oil (3%, group FO) for 110 days. TLR4, TNF-α, and IL-1ß mRNA were ubiquitously expressed throughout the hypothalamic-gonadal axis. However, TLR4 mRNA expression was lower in the hypothalamus than the epididymis (P < 0.01), seminal vesicles (P < 0.01), and pituitary gland (P < 0.05). Dietary enrichment in PUFA did not modify the gene expression profile nor the histological characteristics of the tissues. Conversely in seminal plasma, rabbits fed with L and FO had lower n-6 (P < 0.05), LC-PUFA n-6 (P < 0.05), and n-6/n-3 ratio (P < 0.05) but higher n-3 (P < 0.001) and LC-PUFA n-3 (P < 0.01) compared to the control group. Our study builds a map of the gene expression of TRL4 and proinflammatory cytokines in the reproductive hypothalamic-gonadal axis of the male rabbit, fundamental step for understanding the immune defence mechanisms. Diets enriched in LC-PUFA did not affect basal gene expression but modulated sperm fatty acid composition. Finally, rabbit may be an excellent animal model to study the relationship between inflammation and infertility, and the nutritional modulation of immune functions.

Functional Pituitary Networks in Vertebrates.

The pituitary is a master endocrine gland that developed early in vertebrate evolution and therefore exists in all modern vertebrate classes. The last decade has transformed our view of this key organ. Traditionally, the pituitary has been viewed as a randomly organized collection of cells that respond to hypothalamic stimuli by secreting their content. However, recent studies have established that pituitary cells are organized in tightly wired large-scale networks that communicate with each other in both homo and heterotypic manners, allowing the gland to quickly adapt to changing physiological demands. These networks functionally decode and integrate the hypothalamic and systemic stimuli and serve to optimize the pituitary output into the generation of physiologically meaningful hormone pulses. The development of 3D imaging methods and transgenic models have allowed us to expand the research of functional pituitary networks into several vertebrate classes. Here we review the establishment of pituitary cell networks throughout vertebrate evolution and highlight the main perspectives and future directions needed to decipher the way by which pituitary networks serve to generate hormone pulses in vertebrates.

An Extremely Low Frequency Magnetic Field and Global Cerebral Ischemia Affect Pituitary ACTH and TSH Cells in Gerbils.

The neuroendocrine system can be modulated by a magnetic field and cerebral ischemia as external and internal stressors, respectively. This study deals with the separate or combined effects of an extremely low frequency (ELF) magnetic field (50 Hz, average magnetic field of 0.5 mT) for 7 days and global cerebral ischemia for 10 min on the morpho-functional features of pituitary adrenocorticotrophic (ACTH) and thyrotrophic (TSH) cells in 3-month-old gerbils. To determine the immediate and delayed effects of the applied stressors, measurements were made on the 7th and 14th days after the onset of the experiment. The ELF magnetic field and 10-min global cerebral ischemia, separately and particularly in combination, decreased (P < 0.05) the volume density of ACTH cells, while only in combination were intracellular ACTH content and plasma ACTH concentration increased (P < 0.05) on day 7. The ELF magnetic field elevated serum TSH concentration on day 7 and intracellular TSHß content on day 14 (P < 0.05). Also, 10-min global cerebral ischemia alone increased serum TSH concentration (P < 0.05), while in combination with the ELF magnetic field it elevated (P < 0.05) intracellular TSHß content on day 14. In conclusion, an ELF magnetic field and/or 10-min global cerebral ischemia can induce immediate and delayed stimulation of ACTH and TSH synthesis and secretion. Bioelectromagnetics. 2020;41:91-103. © 2019 Bioelectromagnetics Society.

Characterization of the Inhibitory Effect of Gastrodigenin and Gastrodin on M-type K+ Currents in Pituitary Cells and Hippocampal Neurons.

Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.

Functional survival of rat pituitary gland in hypothermic storage for pituitary transplantation.

PURPOSE: Deteriorated pituitary function can lead to serious complications that might need lifelong hormone replacement therapy. However, long-term hormone administration can have significant adverse effects. Thus, it would be more desirable to restore pituitary function by pituitary transplantation. In this study, we investigated functional preservation of extracted pituitary gland in special preservation solution under hypothermic condition for pituitary transplantation. METHODS: We obtained nineteen pituitary glands from 250-300 g male Sprague-Dawley rats via parapharyngeal approach. These extracted glands were divided into three pieces and stored in histidine-tryptophan-ketoglutarate (HTK) solution at 4 °C and compared to their corresponding glands stored in phosphate buffer saline (PBS). Light and electron microscopic examinations were performed to identify morphological changes of pituitary gland at 0,3, and 7 days after storage. TUNEL assay to confirm cell viability, and adenosine-triphosphate (ATP) concentration were also serially examined. RESULTS: Tissue architecture and cellular viability of specimens preserved in HTK solution for 3 days were considerably maintained and similar to those in normal pituitary gland (0 day specimen). In contrast, specimens stored in PBS were markedly destroyed after 3 days of storage. After 7 days of storage, significant degeneration occurred in tissues stored in both HTK and PBS. However, tissue architecture was preserved more in specimens stored in HTK solution than those stored in PBS. ATP concentration decreased more rapidly in specimens stored in PBS solution, but there was no statistical significance (p= 0.055). CONCLUSIONS: Extracted rat pituitary gland supplemented with special preservation solution could be preserved for 3 days under hypothermic condition.

Soy Phytoestrogens Do Not Fully Reverse Changes in Rat Pituitary Castration Cells: Unbiased Stereological Study.

The aim of the study was to examine the potential of the principal soy isoflavones, genistein and daidzein, or isoflavone rich soy extract to recover pituitary castration cells in orchidectomized adult male rats in comparison with the effects of estradiol. Two weeks post orchidectomy (Orx), animals received estradiol-dipropionate, genistein, daidzein or soy extract subcutaneously for 3 weeks. Control sham-operated (So) and Orx rats received just the vehicle. Changes in the volumes of pars distalis, of individual follicle-stimulating hormone (FSH) and luteinizing hormone (LH) containing cells, their volume, numerical density and number were determined by unbiased design-based stereology. The intracellular content of ßFSH and ßLH was estimated by relative intensity of fluorescence (RIF). Orchidectomy increased all examined stereological parameters and RIF. Compared to Orx, estradiol increased the volume of pars distalis, but reversed RIF and all morphometric parameters of gonadotropes to the level of So rats, except their number. Treatments with purified isoflavones and soy extract decreased RIF to the control So level, expressing an estradiol-like effect. However, the histological appearance and morphometrical features of gonadotropes did not follow this pattern. Genistein increased the volume of pars distalis, decreased the volume density of LH-labeled cells and raised the number of gonadotropes. Daidzein decreased the cell volume of gonadotropic cells but increased their number and numerical density. Soy extract induced an increase in number and numerical density of FSH-containing cells. Therefore, it can be concluded that soy phytoestrogens do not fully reverse the Orx-induced changes in pituitary castration cells. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Signaling pathways to and from the hypophysial pars tuberalis, an important center for the control of seasonal rhythms.

Seasonal (circannual) rhythms play an important role for the control of body functions (reproduction, metabolism, immune responses) in nearly all living organisms. Also humans are affected by the seasons with regard to immune responses and mental functions, the seasonal affective disorder being one of the most prominent examples. The hypophysial pars tuberalis (PT), an important interface between the hypophysial pars distalis and neuroendocrine centers in the brain, plays an essential role in the regulation of seasonal functions and may even be the seat of the circannual clock. Photoperiodic signals provide a major input to the PT. While the perception of these signals involves extraocular photoreceptors in non-mammalian species (birds, fish), mammals perceive photoperiodic signals exclusively in the retina. A multisynaptic pathway connects the retina with the pineal organ where photoperiodic signals are translated into the neurohormone melatonin that is rhythmically produced night by night and encodes the length of the night. Melatonin controls the functional activity of the mammalian PT by acting upon MT1 melatonin receptors. The PT sends its output signals via retrograde and anterograde pathways. The retrograde pathway targetting the hypothalamus employs TSH as messenger and controls a local hypothalamic T3 system. As discovered in Japanese quail, TSH triggers molecular cascades mediating thyroid hormone conversion in the ependymal cell layer of the infundibular recess of the third ventricle. The local accumulation of T3 in the mediobasal hypothalamus (MBH) appears to activate the gonadal axis by affecting the neuro-glial interaction between GnRH terminals and tanycytes in the median eminence. This retrograde pathway is conserved in photoperiodic mammals (sheep and hamsters), and even in non-photoperiodic laboratory mice provided that they are capable to synthesize melatonin. The anterograde pathway is implicated in the control of prolactin secretion, targets cells in the PD and supposedly employs small molecules as signal substances collectively denominated as "tuberalins". Several "tuberalin" candidates have been proposed, such as tachykinins, the secretory protein TAFA and endocannabinoids (EC). The PT-intrinsic EC system was first demonstrated in Syrian hamsters and shown to respond to photoperiodic changes. Subsequently, the EC system was also demonstrated in the PT of mice, rats and humans. To date, 2-arachidonoylglycerol (2-AG) appears as the most important endocannabinoid from the PT. Likely targets for the EC are folliculo-stellate cells that contain the CB1 receptor and appear to contact lactotroph cells. The CB1 receptor was also found on corticotroph cells which appear as a further target of the EC. Recently, the CB1 receptor was also localized to CRF-containing nerve fibers running in the outer zone of the median eminence. This finding suggests that the EC system of the PT contributes not only to the anterograde, but also to the retrograde pathway. Taken together, the results support the concept that the PT transmits its signals via a "cocktail" of messenger molecules which operate also in other brain areas and systems rather than through PT-specific "tuberalins". Furthermore, they may attribute a novel function to the PT, namely the modulation of the stress response and immune functions.

Neuroanatomical distribution and function of the vasopressin V1B receptor in the rat brain deciphered using specific fluorescent ligands.

It is now accepted that vasopressin, through V1A/V1B receptors, centrally regulates cognitive functions such as memory, affiliation, stress, fear and depression. However, the respective roles of these receptor isoforms and their contribution to stress-related pathologies remain uncertain. The development of new therapeutic treatments requires a precise knowledge of the distribution of these receptors within the brain, which has been so far hampered by the lack of selective V1B markers. In the present study, we have determined the pharmacological properties of three new potent rat V1B fluorescent ligands and demonstrated that they constitute valuable tools for simultaneous visualization and activation of native V1B receptors in living rat brain tissue. Thus, d[Leu4,Lys-Alexa 647)8]VP (analogue 3), the compound with the best affinity-selectivity/fluorescence ratio for the V1B receptor emerged as the most promising. The rat brain regions most concerned by stress such as hippocampus, olfactory bulbs, cortex and amygdala display the highest V1B fluorescent labelling with analogue 3. In the hippocampus CA2, V1B receptors are located on glutamatergic, not GABAergic neurones, and are absent from astrocytes. Using AVP-EGFP rats, we demonstrate the presence of V1B autoreceptors on AVP-secreting neurones not only in the hypothalamus, but also sparsely in the hippocampus. Finally, using both electrophysiology and visualization of ERK phosphorylation, we show analogue 3-induced activation of the V1B receptor in situ. This will help to analyse expression and functionality of V1B receptors in the brain and contribute to further explore the AVPergic circuitry in normal and pathological conditions.

Therapeutic effects of Schisandra chinensis on the hyperprolactinemia in rat.

Prolactin (PRL) is secreted from the pituitary gland in response to eating, mating, and ovulation. Increased serum concentration of PRL during pregnancy contributes to enlargement of the mammary glands of the breasts and prepares for production of milk. However, high PRL levels derived from prolactinoma and hyperprolactinemia induce physiological disorders such as infertility and early menopause. Natural compounds isolated from S. chinensis have been known to possess anti-oxidative, anti-inflammatory and anti-diabetic effects. In the present study, we examined the therapeutic effect of S. chinensis and its single compounds on hyperprolactinemia in the pituitary gland. In rat pituitary cells, PRL expression levels were examined using real-time PCR and western blot assay. Crude S. chinensis extract and its single compound, gomisin N, reduced mRNA and protein levels of PRL in GH3 cells. In addition, cell proliferation and PRL target gene expression in cells were modulated by S. chinensis. Similar to the in vitro experiments, crude S. chinensis extract and gomisin N reduced PRL levels in the pituitary and serum of immature female rats. These results show that S. chinensis and its single compound, gomisin N, are regulators of PRL production and may be candidates for treatment of hyperprolactinemia and prolactinoma.

Variable proopiomelanocortin expression in tanycytes of the adult rat hypothalamus and pituitary stalk.

It is generally believed that proopiomelanocortin (POMC) is expressed exclusively by neurons in the adult rodent brain. Unbeknownst to most researchers, however, Pomc in situ hybridization studies in the rat show specific labeling in the ventral wall of the hypothalamic third ventricle, which is formed by specialized ependymal cells, called tanycytes. Here we characterized this non-neuronal POMC expression in detail using in situ hybridization and immunohistochemical techniques, and report two unique characteristics. First, POMC mRNA and precursor protein expression in non-neuronal cells varies to a great degree as to the extent and abundance of expression. In brains with low-level expression, POMC mRNA and protein was largely confined to a population of tanycytes within the infundibular stalk/caudal median eminence, termed here γ tanycytes, and a subset of closely located ß and α2 tanycytes. In brains with high-level expression, POMC mRNA and protein was observed in the vast majority of α2, ß, and γ tanycytes. This variability was observed in both adult males and females; of 41 rats between 8 and 15 weeks of age, 17 had low-, 9 intermediate-, and 15 high-level POMC expression in tanycytes. Second, unlike other known POMC-expressing cells, tanycytes rarely contained detectable levels of adrenocorticotropin or α-melanocyte-stimulating hormone. The results indicate either a dynamic spatiotemporal pattern whereby low and high POMC syntheses in tanycytes occur periodically in each brain, or marked interindividual differences that may persist throughout adulthood. Future studies are required to examine these possibilities and elucidate the physiologic importance of POMC in tanycytes. J. Comp. Neurol. 525:411-441, 2017. © 2016 Wiley Periodicals, Inc.

Research Resource: Real-Time Analysis of Somatostatin and Dopamine Receptor Signaling in Pituitary Cells Using a Fluorescence-Based Membrane Potential Assay.

Stable somatostatin analogues and dopamine receptor agonists are the mainstay for the pharmacological treatment of functional pituitary adenomas; however, only a few cellular assays have been developed to detect receptor activation of novel compounds without disrupting cells to obtain the second messenger content. Here, we adapted a novel fluorescence-based membrane potential assay to characterize receptor signaling in a time-dependent manner. This minimally invasive technique provides a robust and reliable read-out for ligand-induced receptor activation in permanent and primary pituitary cells. The mouse corticotropic cell line AtT-20 endogenously expresses both the somatostatin receptors 2 (sst2) and 5 (sst5). Exposure of wild-type AtT-20 cells to the sst2- and sst5-selective agonists BIM-23120 and BIM-23268, respectively, promoted a pertussis toxin- and tertiapin-Q-sensitive reduction in fluorescent signal intensity, which is indicative of activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. After heterologous expression, sst1, sst3, and sst4 receptors also coupled to GIRK channels in AtT-20 cells. Similar activation of GIRK channels by dopamine required overexpression of dopamine D2 receptors (D2Rs). Interestingly, the presence of D2Rs in AtT-20 cells strongly facilitated GIRK channel activation elicited by the sst2-D2 chimeric ligand BIM-23A760, suggesting a synergistic action of sst2 and D2Rs. Furthermore, stable somatostatin analogues produced strong responses in primary pituitary cultures from wild-type mice; however, in cultures from sst2 receptor-deficient mice, only pasireotide and somatoprim, but not octreotide, induced a reduction in fluorescent signal intensity, suggesting that octreotide mediates its pharmacological action primarily via the sst2 receptor.

Tankyrases regulate glucoregulatory mechanisms and somatic growth via the central melanocortin system in zebrafish larvae.

The central melanocortin system is a key regulator of energy homeostasis. Recent studies indicate that tankyrases (TNKSs), which poly(ADP-ribosyl)ate target proteins and direct them toward proteasomal degradation, affect overall metabolism, but the exact molecular mechanisms remain unclear. We used zebrafish larvae as a model to study the mechanisms by which TNKS1b, the zebrafish ortholog of mammalian TNKS1, regulates glucose homeostasis and somatic growth. In situ hybridization revealed that TNKS1b mRNA is prominently expressed in the hypothalamus and pituitary of the embryonic and larval brain. In the pituitary, TNKS1b is coexpressed with pro-opiomelanocortin a (pomca) gene in corticotropes and melanotropes. Knockdown of TNKS1b reduced the linear growth of the larvae, stimulated insulin gene and glucose transporter 4 protein, and suppressed gluconeogenic phosphoenolpyruvate carboxykinase 1 gene. This result indicates rapid glucose utilization and reduction of gluconeogenesis in TNKS1b-deficient larvae. Knockdown of TNKS1b down-regulated pomca expression and diminished α-melanocyte-stimulating hormone in the pars intermedia. Furthermore, down-regulation of TNKS1b suppressed the expression of melanocortin receptor 3 and increased the expression of melanocortin receptor 4. The collective data suggest that TNKS1b modulates glucoregulatory mechanisms and the somatic growth of zebrafish larvae via the central melanocortin system.

Persistent expression of activated notch in the developing hypothalamus affects survival of pituitary progenitors and alters pituitary structure.

BACKGROUND: As the pituitary gland develops, signals from the hypothalamus are necessary for pituitary induction and expansion. Little is known about the control of cues that regulate early signaling between the two structures. Ligands and receptors of the Notch signaling pathway are found in both the hypothalamus and Rathke's pouch. The downstream Notch effector gene Hes1 is required for proper pituitary formation; however, these effects could be due to the action of Hes1 in the hypothalamus, Rathke's pouch, or both. To determine the contribution of hypothalamic Notch signaling to pituitary organogenesis, we used mice with loss and gain of Notch function within the developing hypothalamus. RESULTS: We demonstrate that loss of Notch signaling by conditional deletion of Rbpj in the hypothalamus does not affect expression of Hes1 within the posterior hypothalamus or expression of Hes5. In contrast, expression of activated Notch within the hypothalamus results in ectopic Hes5 expression and increased Hes1 expression, which is sufficient to disrupt pituitary development and postnatal expansion. CONCLUSIONS: Taken together, our results indicate that Rbpj-dependent Notch signaling within the developing hypothalamus is not necessary for pituitary development, but persistent Notch signaling and ectopic Hes5 expression in hypothalamic progenitors affects pituitary induction and expansion.

Hypophysiotropic gonadotropin-releasing hormone projections are exposed to dense plexuses of kisspeptin, neurokinin B and substance p immunoreactive fibers in the human: a study on tissues from postmenopausal women.

Neuronal populations that synthesize kisspeptin (KP), neurokinin B (NKB) and substance P (SP) in the hypothalamic infundibular nucleus of humans are partly overlapping. These cells are important upstream regulators of gonadotropin-releasing hormone (GnRH) neurosecretion. Homologous neurons in laboratory animals are thought to modulate episodic GnRH secretion primarily via influencing KP receptors on the hypophysiotropic fiber projections of GnRH neurons. To explore the structural basis of this putative axo-axonal communication in humans, we analyzed the anatomical relationship of KP-immunoreactive (IR), NKB-IR and SP-IR axon plexuses with hypophysiotropic GnRH fiber projections. Immunohistochemical studies were carried out on histological samples from postmenopausal women. The neuropeptide-IR axons innervated densely the portal capillary network in the postinfundibular eminence. Subsets of the fibers formed descending tracts in the infundibular stalk, some reaching the neurohypophysis. KP-IR, NKB-IR and SP-IR plexuses intermingled, and established occasional contacts, with hypophysiotropic GnRH fibers in the postinfundibular eminence and through their lengthy course while descending within the infundibular stalk. Triple-immunofluorescent studies also revealed considerable overlap between the KP, NKB and SP signals in individual fibers, providing evidence that these peptidergic projections arise from neurons of the mediobasal hypothalamus. These neuroanatomical observations indicate that the hypophysiotropic projections of human GnRH neurons in the postinfundibular eminence and the descending GnRH tract coursing through the infundibular stalk to the neurohypophysis are exposed to neurotransmitters/neuropeptides released by dense KP-IR, NKB-IR and SP-IR fiber plexuses. Localization and characterization of axonal neuropeptide receptors will be required to clarify the putative autocrine and paracrine interactions in these anatomical regions.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells.

BACKGROUND: Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS: In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS: Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION: Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

A novel neuropeptide in suppressing luteinizing hormone release in goldfish, Carassius auratus.

The fish reproductive axis is regulated by many neuroendocrine factors. However, factors involved in the suppression of this axis are largely uncharacterized. In this study, we describe a novel neuropeptide derived from the spexin precursor acting as a negative factor to suppress the reproductive axis in teleost. The cDNA sequences of the spexin precursors have been cloned from both zebrafish and goldfish. A 14-aa mature peptide with the C-terminal amidated (spexin-14a: NWTPQAMLYLKGTQ-NH2) is conceivably generated by processing of the spexin precursors in both species. Spexin is mainly expressed in the brain and ovary of zebrafish and spexin-14a-ir cells are located in several brain regions of goldfish. Functionally, goldfish spexin-14a could significantly suppress luteinizing hormone (LH) release in cultured goldfish pituitary cells. Moreover, intraperitoneal injection of spexin-14a could effectively suppress serum LH level. The mRNA expression of spexin is lower in the breeding season and hypothalamic expression of spexin is regulated by gonadal hormones. These results constitute the first report on the novel role of spexin in the negative regulation of the reproductive axis in teleost.

Coordination of hypothalamic and pituitary T3 production regulates TSH expression.

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3'-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus.

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHß mRNA levels. Only GnIH-II decreased LHß mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHß and FSHß synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.